Novel nucleoside triphosphate transporter and uses thereof

Abstract

Disclosed herein are proteins, methods, cells, engineered microorganisms, and kits for generating a modified nucleoside triphosphate transporter from Phaeodactylum tricornutum . Also disclosed herein proteins, methods, cells, engineered microorganisms, and kits for production of a nucleic acid molecule that comprises an unnatural nucleotide utilizing a modified nucleoside triphosphate transporter from Phaeodactylum tricornutum.

What is claimed is: 
     
         1 . An isolated and modified nucleoside triphosphate transporter from  Phaeodactylum tricornutum  (PtNTT2) comprising a deletion, wherein the isolated and modified nucleoside triphosphate transporter is obtained from an engineered cell. 
     
     
         2 . The isolated and modified nucleoside triphosphate transporter of  claim 1 , wherein the deletion is a terminal deletion or an internal deletion. 
     
     
         3 . The isolated and modified nucleoside triphosphate transporter of  claim 2 , wherein the deletion is a terminal deletion. 
     
     
         4 . The isolated and modified nucleoside triphosphate transporter of  claim 2 , wherein the deletion is an internal deletion. 
     
     
         5 . The isolated and modified nucleoside triphosphate transporter of  claim 3 , wherein the terminal deletion is an N-terminal deletion, a C-terminal deletion, or a deletion of both termini. 
     
     
         6 . The isolated and modified nucleoside triphosphate transporter of  claim 3 , wherein the terminal deletion is an N-terminal deletion. 
     
     
         7 . The isolated and modified nucleoside triphosphate transporter of  claim 2 , wherein the deletion comprises about 5, 10, 15, 20, 22, 25, 30, 40, 44, 50, 60, 66, 70, or more amino acid residues. 
     
     
         8 . The isolated and modified nucleoside triphosphate transporter of  claim 2 , wherein the isolated and modified nucleoside triphosphate transporter comprises a deletion of about 5, 10, 15, 20, 22, 25, 30, 40, 44, 50, 60, 66, 70, or more amino acid residues at the N-terminus. 
     
     
         9 . The isolated and modified nucleoside triphosphate transporter of  claim 8 , wherein the isolated and modified nucleoside triphosphate transporter comprises a deletion of about 66 amino acid residues at the N-terminus. 
     
     
         10 . The isolated and modified nucleoside triphosphate transporter of  claim 1 , wherein the isolated and modified nucleoside triphosphate transporter comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity sequence identity to SEQ ID NO: 4. 
     
     
         11 . The isolated and modified nucleoside triphosphate transporter of  claim 1 , wherein the isolated and modified nucleoside triphosphate transporter comprises 100% sequence identity to SEQ ID NO: 4. 
     
     
         12 . The isolated and modified nucleoside triphosphate transporter of  claim 1 , wherein the isolated and modified nucleoside triphosphate transporter comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6. 
     
     
         13 . The isolated and modified nucleoside triphosphate transporter of  claim 1 , wherein the isolated and modified nucleoside triphosphate transporter comprises 100% sequence identity to SEQ ID NO: 6. 
     
     
         14 . The isolated and modified nucleoside triphosphate transporter of  claim 1 , wherein the isolated and modified nucleoside triphosphate transporter comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. 
     
     
         15 . The isolated and modified nucleoside triphosphate transporter of  claim 1 , wherein the isolated and modified nucleoside triphosphate transporter comprises 100% sequence identity to SEQ ID NO: 8. 
     
     
         16 . The isolated and modified nucleoside triphosphate transporter of  claim 1 , wherein the isolated and modified nucleoside triphosphate transporter further comprises a signal peptide. 
     
     
         17 . The isolated and modified nucleoside triphosphate transporter of  claim 16 , wherein the signal peptide is selected from Table 3. 
     
     
         18 . The isolated and modified nucleoside triphosphate transporter of  claim 1 , wherein the engineered cell comprises a prokaryotic cell. 
     
     
         19 . The isolated and modified nucleoside triphosphate transporter of  claim 1 , wherein the engineered cell is  E. coli.    
     
     
         20 . A nucleic acid molecule encoding an isolated and modified nucleoside triphosphate transporter of  claims 1 - 19 . 
     
     
         21 . Use of a modified nucleoside triphosphate transporter of  claims 1 - 19  for the incorporation of an unnatural triphosphate during the synthesis of a nucleic acid molecule. 
     
     
         22 . An engineered cell comprising:
 a first nucleic acid molecule encoding a modified nucleoside triphosphate transporter from  Phaeodactylum tricornutum  (PtNTT2).   
     
     
         23 . The engineered cell of  claim 22 , wherein the nucleic acid of the modified nucleoside triphosphate transporter is incorporated in the genomic sequence of the engineered cell. 
     
     
         24 . The engineered cell of  claim 22 , wherein the engineered cell comprises a plasmid comprising the modified nucleoside triphosphate transporter. 
     
     
         25 . The engineered cell of  claim 22 , wherein the modified nucleoside triphosphate transporter is a codon optimized nucleoside triphosphate transporter from  Phaeodactylum tricornutum.    
     
     
         26 . The engineered cell of  claim 22 , wherein the modified nucleoside triphosphate transporter comprises a deletion. 
     
     
         27 . The engineered cell of  claim 26 , wherein the deletion is a terminal deletion or an internal deletion. 
     
     
         28 . The engineered cell of  claim 27 , wherein the deletion is an N-terminal truncation, a C-terminal truncation, or a truncation of both termini. 
     
     
         29 . The engineered cell of  claim 26 , wherein the modified nucleoside triphosphate transporter comprises a deletion of about 5, 10, 15, 20, 22, 25, 30, 40, 44, 50, 60, 66, 70, or more amino acid residues. 
     
     
         30 . The engineered cell of  claim 26 , wherein the modified nucleoside triphosphate transporter comprises a deletion of about 5, 10, 15, 20, 22, 25, 30, 40, 44, 50, 60, 66, 70, or more amino acid residues at the N-terminus. 
     
     
         31 . The engineered cell of  claim 30 , wherein the modified nucleoside triphosphate transporter comprises a deletion of about 66 amino acid residues at the N-terminus. 
     
     
         32 . The engineered cell of  claim 22 , wherein the isolated and modified nucleoside triphosphate transporter comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity sequence identity to SEQ ID NO: 4. 
     
     
         33 . The engineered cell of  claim 22 , wherein the isolated and modified nucleoside triphosphate transporter comprises 100% sequence identity to SEQ ID NO: 4. 
     
     
         34 . The engineered cell of  claim 22 , wherein the isolated and modified nucleoside triphosphate transporter comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6. 
     
     
         35 . The engineered cell of  claim 22 , wherein the isolated and modified nucleoside triphosphate transporter comprises 100% sequence identity to SEQ ID NO: 6. 
     
     
         36 . The engineered cell of  claim 22 , wherein the isolated and modified nucleoside triphosphate transporter comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. 
     
     
         37 . The engineered cell of  claim 22 , wherein the isolated and modified nucleoside triphosphate transporter comprises 100% sequence identity to SEQ ID NO: 8. 
     
     
         38 . The engineered cell of  claim 22 , wherein the modified nucleoside triphosphate transporter is under the control of a promoter selected from an  E. coli  promoter or a phage promoter. 
     
     
         39 . The engineered cell of  claim 38 , wherein the promoter is selected from P bla , P lac , P lacUV5 , P H207 , P λ , P tac , or P N25 . 
     
     
         40 . The engineered cell of  claim 38 , wherein the modified nucleoside triphosphate transporter is under the control of promoter P lacUV5 . 
     
     
         41 . The engineered cell of  claim 38 , wherein the modified nucleoside triphosphate transporter is under the control of a promoter from a lac operon. 
     
     
         42 . The engineered cell of  claim 22 , wherein the modified nucleoside triphosphate transporter is encoded within a pSC plasmid. 
     
     
         43 . The engineered cell of  claim 22 , wherein the modified nucleoside triphosphate transporter decreases doubling time of the engineered cell. 
     
     
         44 . The engineered cell of  claim 22 , wherein the modified nucleoside triphosphate transporter enables unnatural base pair retention of about 50%, 60%, 70%, 80%, 90%, 95%, 99% or more. 
     
     
         45 . The engineered cell of  claim 22 , wherein the engineered cell further comprises a second nucleic acid molecule encoding a Cas9 polypeptide or variants thereof, a third nucleic acid molecule encoding a single guide RNA (sgRNA) comprising a crRNA-tracrRNA scaffold; and a fourth nucleic acid molecule comprising an unnatural nucleotide. 
     
     
         46 . The engineered cell of  claim 45 , wherein the second nucleic acid molecule, the third nucleic acid molecule, and the fourth nucleic acid molecule are encoded in one or more plasmids. 
     
     
         47 . The engineered cell of  claim 46 , wherein the sgRNA encoded by the third nucleic acid molecule comprises a target motif that recognizes a modification at the unnatural nucleotide position within the fourth nucleic acid molecule. 
     
     
         48 . The engineered cell of  claim 47 , wherein the modification at the unnatural nucleotide position within the third nucleic acid molecule generates a modified third nucleic acid molecule. 
     
     
         49 . The engineered cell of  claim 47 , wherein the modification is a substitution. 
     
     
         50 . The engineered cell of  claim 47 , wherein the modification is a deletion. 
     
     
         51 . The engineered cell of  claim 47 , wherein the modification is an insertion. 
     
     
         52 . The engineered cell of  claim 45 , wherein the sgRNA encoded by the third nucleic acid molecule further comprises a protospacer adjacent motif (PAM) recognition element. 
     
     
         53 . The engineered cell of  claim 52 , wherein the PAM element is adjacent to the 3′ terminus of the target motif. 
     
     
         54 . The engineered cell of  claim 45 , wherein the combination of Cas9 polypeptide or variants thereof and sgRNA modulates replication of the modified fourth nucleic acid molecule. 
     
     
         55 . The engineered cell of  claim 45 , wherein the combination of Cas9 polypeptide or variants thereof, sgRNA and the modified nucleoside triphosphate transporter modulates replication of the modified fourth nucleic acid molecule. 
     
     
         56 . The engineered cell of  claim 45 , wherein the combination of Cas9 polypeptide or variants thereof, sgRNA and the modified nucleoside triphosphate transporter decreases the replication rate of the modified fourth nucleic acid molecule by about 80%, 85%, 95%, 99%, or higher. 
     
     
         57 . The engineered cell of  claim 45 , wherein the production of the fourth nucleic acid molecule in the engineered cell increases by about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or higher. 
     
     
         58 . The engineered cell of  claim 45 , wherein the Cas9 polypeptide or variants thereof generate a double-stranded break. 
     
     
         59 . The engineered cell of  claim 45 , wherein the Cas9 polypeptide is a wild-type Cas9. 
     
     
         60 . The engineered cell of  claim 22 , wherein the unnatural nucleotide comprises an unnatural base selected from the group consisting of 2-aminoadenin-9-yl, 2-aminoadenine, 2-F-adenine, 2-thiouracil, 2-thio-thymine, 2-thiocytosine, 2-propyl and alkyl derivatives of adenine and guanine, 2-amino-adenine, 2-amino-propyl-adenine, 2-aminopyridine, 2-pyridone, 2′-deoxyuridine, 2-amino-2′-deoxyadenosine 3-deazaguanine, 3-deazaadenine, 4-thio-uracil, 4-thio-thymine, uracil-5-yl, hypoxanthin-9-yl (I), 5-methyl-cytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 5-bromo, and 5-trifluoromethyl uracils and cytosines; 5-halouracil, 5-halocytosine, 5-propynyl-uracil, 5-propynyl cytosine, 5-uracil, 5-substituted, 5-halo, 5-substituted pyrimidines, 5-hydroxycytosine, 5-bromocytosine, 5-bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 6-alkyl derivatives of adenine and guanine, 6-azapyrimidines, 6-azo-uracil, 6-azo cytosine, azacytosine, 6-azo-thymine, 6-thio-guanine, 7-methylguanine, 7-methyladenine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-adenine, 7-deaza-8-azaguanine, 8-azaguanine, 8-azaadenine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, and 8-hydroxyl substituted adenines and guanines; N4-ethylcytosine, N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, tricyclic pyrimidines, phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps, phenoxazine cytidine (9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido [3′,2′:4,5]pyrrolo [2,3-d]pyrimidin-2-one), 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methythio-N6-isopentenyladeninje, uracil-5-oxyacetic acid, wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxacetic acid methylester, uracil-5-oxacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine and those in which the purine or pyrimidine base is replaced with a heterocycle. 
     
     
         61 . The engineered cell of  claim 60 , wherein the unnatural base is selected from the group consisting of 
       
         
           
           
               
               
           
         
       
     
     
         62 . The engineered cell of  claim 22 , wherein the unnatural nucleotide further comprises an unnatural sugar moiety. 
     
     
         63 . The engineered cell of  claim 62 , wherein the unnatural sugar moiety is selected from the group consisting of a modification at the 2′ position: OH; substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2  CH 3 , ONO 2 , NO 2 , N 3 , NH 2 F; O-alkyl, S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; O-alkyl-O-alkyl, 2′-F, 2′-OCH 3 , 2′-O(CH 2 ) 2 OCH 3  wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 -C 10 , alkyl, C 2 -C 10  alkenyl, C 2 -C 10  alkynyl, —O[(CH2)n O]mCH 3 , —O(CH 2 )nOCH 3 , —O(CH 2 )n NH 2 , —O(CH 2 )n CH 3 , —O(CH 2 )n-ONH 2 , and —O(CH 2 )nON[(CH 2 )n CH 3 )] 2 , where n and m are from 1 to about 10; and/or a modification at the 5′ position: 5′-vinyl, 5′-methyl (R or S), a modification at the 4′ position, 4′-S, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and any combination thereof. 
     
     
         64 . The engineered cell of  claim 22 , wherein the unnatural nucleotide further comprises an unnatural backbone. 
     
     
         65 . The engineered cell of  claim 64 , wherein the unnatural backbone is selected from the group consisting of a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, C 1 -C 10  phosphonates, 3′-alkylene phosphonate, chiral phosphonates, phosphinates, phosphoramidates, 3′-amino phosphoramidate, aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. 
     
     
         66 . The engineered cell of  claim 45 , wherein the sgRNA has less than about 20%, 15%, 10%, 5%, 3%, 1%, or less off-target binding rate. 
     
     
         67 . The engineered cell of  claim 45 , further comprising an additional nucleic acid molecule that encodes an additional single guide RNA (sgRNA) comprising a crRNA-tracrRNA scaffold. 
     
     
         68 . The engineered cell of  claim 22 , wherein the engineered cell is a semi-synthetic organism. 
     
     
         69 . An in vivo method of increasing the production of a nucleic acid molecule containing an unnatural nucleotide comprising an engineered cell of  claims 22 - 68 . 
     
     
         70 . A nucleic acid molecule containing an unnatural nucleotide produced by an engineered cell of  claims 22 - 68 . 
     
     
         71 . An isolated and purified plasmid comprising:
 a nucleic acid molecule encoding a modified nucleoside triphosphate transporter from  Phaeodactylum tricornutum  (PtNTT2); and   a promoter region selected from a pSC plasmid or lacZYA locus.   
     
     
         72 . The isolated and purified plasmid of  claim 71 , wherein the modified nucleoside triphosphate transporter is a codon optimized nucleoside triphosphate transporter from  Phaeodactylum tricornutum.    
     
     
         73 . The isolated and purified plasmid of  claim 71 , wherein the modified nucleoside triphosphate transporter comprises a deletion. 
     
     
         74 . The isolated and purified plasmid of  claim 73 , wherein the deletion is a terminal deletion or an internal deletion. 
     
     
         75 . The isolated and purified plasmid of  claim 74 , wherein the deletion is an N-terminal truncation, a C-terminal truncation, or a truncation of both termini. 
     
     
         76 . The isolated and purified plasmid of  claim 73 , wherein the modified nucleoside triphosphate transporter comprises a deletion of about 5, 10, 15, 20, 22, 25, 30, 40, 44, 50, 60, 66, 70, or more amino acid residues. 
     
     
         77 . The isolated and purified plasmid of  claim 73 , wherein the modified nucleoside triphosphate transporter comprises a deletion of about 5, 10, 15, 20, 22, 25, 30, 40, 44, 50, 60, 66, 70, or more amino acid residues at the N-terminus. 
     
     
         78 . The isolated and purified plasmid of  claim 77 , wherein the modified nucleoside triphosphate transporter comprises a deletion of about 66 amino acid residues at the N-terminus. 
     
     
         79 . The isolated and purified plasmid of  claim 71 , wherein the isolated and modified nucleoside triphosphate transporter comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity sequence identity to SEQ ID NO: 4. 
     
     
         80 . The isolated and purified plasmid of  claim 71 , wherein the isolated and modified nucleoside triphosphate transporter comprises 100% sequence identity to SEQ ID NO: 4. 
     
     
         81 . The isolated and purified plasmid of  claim 71 , wherein the isolated and modified nucleoside triphosphate transporter comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6. 
     
     
         82 . The isolated and purified plasmid of  claim 71 , wherein the isolated and modified nucleoside triphosphate transporter comprises 100% sequence identity to SEQ ID NO: 6. 
     
     
         83 . The isolated and purified plasmid of  claim 71 , wherein the isolated and modified nucleoside triphosphate transporter comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. 
     
     
         84 . The isolated and purified plasmid of  claim 71 , wherein the isolated and modified nucleoside triphosphate transporter comprises 100% sequence identity to SEQ ID NO: 8. 
     
     
         85 . The isolated and purified plasmid of  claim 71 , wherein the promoter region is selected from P bla , P lac , P lacUV5 , P H207 , P λ , P tac , or P N25 . 
     
     
         86 . The isolated and purified plasmid of  claim 71 , wherein the promoter region is selected from P lacI , P bla , or P lac . 
     
     
         87 . The isolated and purified plasmid of  claim 71 , wherein the plasmid is a prokaryotic plasmid. 
     
     
         88 . An in vivo method of increasing the production of a nucleic acid molecule containing an unnatural nucleotide comprising incubating a cell with an isolated and purified plasmid of  claims 71 - 87 . 
     
     
         89 . A kit comprising an isolated and modified nucleoside triphosphate transporter of  claims 1 - 19 . 
     
     
         90 . A kit comprising an engineered cell of  claims 22 - 68 . 
     
     
         91 . A kit comprising an isolated and purified plasmid of  claims 71 - 87 .